The present invention relates to cationic lipids which are used to enhance delivery of biologically active agents, particularly polynucleotides, proteins, peptides, and drug molecules, by facilitating transmembrane transport or by encouraging adhesion to biological surfaces. It relates particularly to cationic lipids comprising ammonium groups.
Some bioactive substances do not need to enter cells to exert their biological effect, because they operate either by acting on cell surfaces through cell surface receptors or to by interacting with extracellular components. However, many natural biological molecules and their analogues, including proteins and polynucleotides, or foreign substances, such as drugs, which are capable of influencing cell function at the subcellular or molecular level are preferably incorporated within the cell in order to produce their effect. For these agents the cell membrane presents a selective barrier which is impermeable to them.
Just as the plasma membrane of a cell is a selective barrier preventing random introduction of potentially toxic substances into the cell, the human body is surrounded by protective membranes which serve a similar defensive function to the whole organism. These membranes include skin, gastric mucosa, nasal mucosa and the like. While these membranes serve a protective function preventing entry of toxic substances, they can also prevent passage of potentially beneficial therapeutic substances into the body. The complex composition of the cell membrane comprises phospholipids, glycolipids, and cholesterol, as well as intrinsic and extrinsic proteins, and its functions are influenced by cytoplasmic components which include Ca.sup.++ and other metal ions, anions, ATP, microfilaments, microtubules, enzymes, and Ca.sup.++ -binding proteins. Interactions among structural and cytoplasmic cell components and their response to external signals make up transport processes responsible for the membrane selectivity exhibited within and among cell types.
Successful intracellular delivery of agents not naturally taken up by cells has been achieved by exploiting the natural process of intracellular membrane fusion, or by direct access of the cell's natural transport mechanisms which include endocytosis and pinocytosis (Duzgunes, N., Subcellular Biochemistry 11:195-286 (1985).
The membrane barrier can be overcome in the first instance by associating these substances in complexes with lipid formulations closely resembling the lipid composition of natural cell membranes. These lipids are able to fuse with the cell membranes on contact, and in the process, the associated substances are delivered intracellularly. Lipid complexes can not only facilitate intracellular transfers by fusing with cell membranes but also by overcoming charge repulsions between the cell membrane and the molecule to be inserted. The lipids of the formulations comprise an amphipathic lipid, such as the phospholipids of cell membranes, and form hollow lipid vesicles, or liposomes, in aqueous systems. This property can be used to entrap the substance to be delivered within the liposomes; in other applications, the drug molecule of interest can be incorporated into the lipid vesicle as an intrinsic membrane component, rather than entrapped into the hollow aqueous interior.
Intracellular delivery of beneficial or interesting proteins can be achieved by introducing expressible DNA and mRNA into the cells of a mammal, a useful technique termed transfection. Gene sequences introduced in this way can produce the corresponding protein coded for by the gene by using endogenous protein synthetic enzymes. The therapy of many diseases could be enhanced by the induced intracellular production of peptides which could remain inside the target cell, be secreted into the local environment of the target cell, or be secreted into the systemic circulation to produce their effect.
Various techniques for introducing the DNA or mRNA precursors of bioactive peptides into cells include the use of viral vectors, including recombinant vectors and retroviruses, which have the inherent ability to penetrate cell membranes. However, the use of such viral agents to integrate exogenous DNA into the chromosomal material of the cell carries a risk of damage to the genome and the possibility of inducing malignant transformation. Another aspect of this approach which restricts its use in vivo is that the integration of DNA into the genome accomplished by these methods implies a loss of control over the expression of the peptide it codes for, so that transitory therapy is difficult to achieve and potential unwanted side effects of the treatment could be difficult or impossible to reverse or halt.
Liposomes have been discussed as possible in vivo delivery vehicles and some encouraging results using this approach to the intracellular expression of DNA have been obtained (Mannino, R. J. Fould-Fogerite, S., Biotechniques 6, 682-690 (1988); Itani, T., Ariga, H., Yamaguchi, N., Tadakuma, T. & Yasuda, T. Gene 56 267-276 (1987); Nicolau, C. Legrand, A. & Grosse, G. E. Meth. Enz. 149 157-176 (1987); Straubinger, R. M. & Papahadjopoulos, D. Meth. Enz. 101 512-527 (1983); Wang, C. Y. & Huang, L. Proc Natl. Acad. Sci. USA 84 7851-7855 (1987)); however, the methodology has fundamental problems. Chief among the difficulties is the failure of liposomes to fuse with the target cell surface, but to be taken up phagocytically instead. Phagocytized liposomes are delivered to the lysosomal compartment, where polynucleotides are subjected to the action of digestive enzymes and degraded, leading to low efficiency of expression.
A major advance in this area was the discovery that a positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), in the form of liposomes, or small vesicles, could interact spontaneously with DNA to form lipid-DNA complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in both uptake and expression of the DNA (Felgner, P. L. et al. Proc. Natl. Acad. Sci., USA 84:7413-7417 (1987) and U.S. Pat. No. 4,897,355 to Eppstein, D. et al.). Others have successfully used a DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP) in combination with a phospholipid to form DNA-complexing vesicles. The Lipofectin.TM. reagent (Bethesda Research Laboratories, Gaithersburg, Md.), an effective agent for the delivery of highly anionic polynucleotides into living tissue culture cells comprises positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional polynucleotide into, for example, tissue culture cells.
Although the use of known cationic lipids overcomes many problems associated with conventional liposome technology for polynucleotide delivery in vitro, several problems related to both in vitro and in vivo applications remain. First, although the efficiency of cationic lipid mediated delivery is relatively high compared to other methods, the absolute level of gene product produced is typically only several hundred copies per cell on average. Thus it would be desirable to improve delivery and expression by a factor of 10 to 1000-fold to achieve useful methodologies. Secondly, known cationic lipids such as DOTMA are toxic to tissue culture cells; thus, any improvements that reduce in vitro toxicity would strengthen the methodology.
A significant body of information is emerging regarding the use of other cationic lipids for the delivery of macromolecules into cells. Loyter prepared vesicles containing a quaternary ammonium surfactant that are capable of transferring functional tobacco mosaic virus into plant protoplasts. (Ballas, N., Zakai, N., Sela, I. and Loyter, A. Biochim. Biophys Acta 939 8-18 (1988)). Huang used cetyltrimethylammonium bromide to obtain functional expression from the chloramphenicol acetyl transferase gene transfected into mouse fibroblasts (Pinnaduwage, P., Schmitt, L. and Huang, L. Biochim. Biophys Acta 985 33-37 (1989)). Behr has shown that a novel lipophilic derivative of spermine can transfect primary pituitary cells (Behr, J-P, Demeneix, B., Loeffler, J-P and Perez-Mutul, J. Proc. Natl. Acad. Sci. USA 86 6982-6986 (1989)). Finally, John Silvius has shown that a cationic lipid (DOTAP), originally synthesized by Eibl (Eibl, H. and Woolley, P. Biophys. Chem. 10 261-271 (1979)) forms liposomes which can- fuse with negatively charged liposomes and can deliver functional DNA and RNA into tissue culture fibroblasts (Stamatatos, L., Leventis, R., Zuckermann, M. J. & Silvius, J. R. Biochemistry 27 3917-3925 (1988)). Other laboratories have studies the physical properties of vesicles formed from synthetic cationic amphophiles (Rupert, L. A. M., Hoekstra, D. and Engberts, J. B. F. N. Am. Chem. Soc. 108: 2628-2631 (1985); Carmona-Ribeiro, A. M., Yoshida, L. S. and Chaimovich, H. J. Phys Chem 89 2928-2933 (1985); Rupert, L. A. M., Engberts, J. B. F. N. and Hoekstra, D. J. Amer. Chem. Soc. 108:3920-3925 (1986)).
It is not feasible to extend in vitro transfection technology to in vivo applications directly. In vivo, the diether lipids, such as DOTMA or Lipofectin the current commercial standard, would be expected to accumulate in the body due to the poorly metabolized ether bonds. And finally, it has been reported that the cationic lipid transfection methodology is inhibited by serum; for in vivo applications conditions must be identified which allow transfection to occur in a complex biological milieu such as 100% serum.
Therefore, while the known lipofection technique of transfection described is more efficient and satisfactory than previously known procedures, and permits transient as well as stable transfection and peptide expression, it is not understood what factors regulate the efficiency of the transfection process and how it may be optimized. It would be desirable to determine these factors in order to develop an intracellular delivery system having the advantages of the above-described systems but without their inherent limitations.
Accordingly, it is an object of the invention to provide cationic lipids which carry out both stable and transient transfections of polynucleotides such as DNA and mRNA into cells more effectively.
It is also an object of the invention to provide cationic lipids which deliver other molecules of therapeutic interest, including proteins, peptides and small organic molecules, into cells more effectively.
Further, it is an object of the invention to provide cationic lipids that are not only more effective in accomplishing intracellular delivery but are also metabolizable so as to have reduced in vivo and in vitro toxicity.
It is another object of the invention to provide transfection formulation, comprising novel cationic lipids, that are optimally effective in both in vivo and in vitro transfection.